2-amino-9-[(2-hydroxymethyl) cyclopropylidenemethyl] purine antiviral agents

ABSTRACT

Compounds which are active against viruses have the following Formulas: 
     
       
         
         
             
             
         
       
         
         
           
             wherein B is 2-aminopurine-9-yl, which may be unsubstituted or substituted in the 6 position with NHR 1 , OR 2 , or SR 3 ; 
             R 1  is selected from the group consisting of alkyl, alkenyl, alkynyl, and C 4-18  cycloalkyl, any of which may be optionally substituted with one or more members of the group consisting of hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl and aryl; 
             R 2  is selected from the group consisting of C 2-18  alkyl, alkenyl, alkynyl, and cycloalkyl, any of which may be branched or unbranched and optionally substituted with one or more members of the group consisting of hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl, and aryl; and 
             R 3  is selected from the group consisting of alkyl, alkenyl, alkynyl, and cycloalkyl, any of which may be branched or unbranched and optionally substituted with one or more members of the group consisting of hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl and aryl.

RELATED APPLICATIONS

This patent application is a continuation of co-pending InternationalApplication No. PCT/US03/007909, filed Mar. 13, 2003, which claims thebenefit of Provisional Application No. 60/364,495, filed Mar. 15, 2002,which is incorporated herein by reference.

SPONSORSHIP

Work on this invention was supported in part by the National Instituteof Allergy and Infectious Diseases, Grant Nos. U19-AI31718 andP01-AI46390. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to novel 2-aminopurines havingantiviral activity and methods for making and using them.

BACKGROUND OF THE INVENTION

Viruses are the etiologic cause of many life-threatening human diseases.Of special importance are herpes viruses such as herpes simplex 1(HSV-1), herpes simplex 2 (HSV-2), cytomegalovirus (CMV), Epstein-Barrvirus (EBV), varicella zoster virus (VZV) and human herpes viruses 6, 7and 8 (HHV-6, -7 and -8) which are associated with many common viralillnesses. The HSV-1 and HSV-2 infections are characterized by coldsores of skin, mouth or genital region. After primary infection, thevirus is harbored in neural cells and can reappear later in the life ofa patient. Human CMV (HCMV) infection is a life-long affliction whichcan result in morbidity and mortality. These pathologies includemicrocephaly, hepatosplenomegaly, jaundice, encephalitis, infections ofthe newborn infants or fetuses in utero, and infections ofimmuno-compromised hosts. HCMV infection is responsible for retinitis,gastritis and pneumonitis in AIDS patients and HCMV-induced pneumoniasor hepatitis are frequent and serious complications of organ or bonemarrow transplants. EBV causes infectious mononucleosis and it isconsidered as the etiologic agent of nasopharyngeal cancer,immunoblastic lymphoma, Burkitt's lymphoma and hairy leukoplakia. VZVcauses chicken pox and shingles. Although in children the chicken pox isusually a non-fatal disease, the recurrent form of this infection,shingles, may in advanced stage lead to paralysis, convulsions andultimately death. Again, in immunocompromised patients the infectionwith VZV is a serious complication. Human herpes virus 6 (HHV-6) whichis commonly associated with children's rash was also identified inacquired immunodeficiency syndrome (AIDS) patients and it may be acofactor in the pathogenesis of AIDS in hosts infected with humanimmunodeficiency virus (HIV). Levine, A. J. Viruses, Ch. 4, W. H.Freeman & Co., New York, pp. 67–85 (1992); Human Herpesvirus Infections,Raven Press, New York (1986); Schirmer, E. C., et al., Proc. Natl. Acad.Sci. USA 88:5922–5926 (1992). Human herpes virus 8 (HHV-8) wasidentified in patients with Kaposi sarcoma, a fatal afflictionaccompanying AIDS. Chang, Y., et al. Science 266:1865–1869 (1994).

HIV is the underlying cause of AIDS, a world-wide epidemic with fatalconsequences. According to the Joint United Nations Programme onHIV/AIDS, 40 million people are estimated to be living with HIV/AIDS atthe end of 2001. During that same year, AIDS caused the deaths of anestimated 3 million people.

Hepatitis B virus (HBV) is a virus that causes chronic diseaseresponsible for serious liver damage, including cirrhosis of the liver,cancer, organ failure and ultimately, death. It is estimated thatapproximately 300 million people worldwide are infected with HBV.According to the CDC, there are approximately 1.25 million Americanschronically infected with HBV. Although use of a prophylactic vaccinehas reduced the incidence of new HBV infections, there continues to be aneed for an effective therapeutic drug.

Various derivatives of nucleoside analogues have been found to exhibitantiviral activity. Notably, acyclovir (Zovirax) and and its prodrugvalacyclovir (Valtrex) are approved drugs for infections caused by HSV-1and HSV-2. Acyclovir Therapy for Herpesvirus Infections (Baker, Ed.), M.Dekker, New York (1990); Against HCMV, four drugs are currentlyavailable: Ganciclovir (Cytovene), cidofovir (Vistide), antisenseoligonucleotide fomivirsen (Vitravene) and foscarnet (Foscavir).However, only ganciclovir is effective orally but it requires largedoses and produces potentially serious adverse effects such as bonemarrow suppression. Ganciclovir Therapy for Cytomegalovirus Infection(Spector, S. S., Ed.), M. Dekker, New York (1991). A considerable effortwent into design, synthesis and biological investigation of analogues ofthese drugs as well as in development of new antiviral agents. Larsson,A., et al., Antimicrob. Agents & Chemother. 30:598–605 (1986); Ashton,W. T., et al., J. Med. Chem. 31:2304–2315 (1988). Cidofovir andfomivirsen are approved only for topical application against retinitisin AIDS patients and foscamet is used only by intravenous route and itleads to characteristic toxicity.

Current drugs for AIDS include AZT (zidovudine, Retrovir), ddI(didanosine, Videx), ddC (zalcitabine, Hivid) and d4T (stavudine,Zerit). De Clercq, E., J. Med. Chem. 38:2491–2517 (1995). Allenicnucleoside analogues such as adenallene and cytallene are examples ofanti-HIV agents containing an unsaturated alkyl group. U.S. Pat.No.4,935,427; Zemlicka, J., Allenols Derived from Nucleic Acid Bases—aNew Class of Anti-HIV Agents: Chemistry and Biological Activity inNucleosides and Nucleotides as Antitumor and Antiviral Agents (Chu, C.K.; Baker, D. C., Eds.), Plenum Press, New York, pp. 73–100 (1993). ForHBV, alpha interferon and 3TC (lamivudine; Epivir) are two drugslicensed for the treatment of persons with chronic HBV infection.Unfortunately, only about 40% of patients respond to these drugs andresistance is a growing problem.

Particular 2-hydroxymethylcyclopropylidenemethylpurines and theirutility against certain viruses have been described elsewhere (see, forexample, co-owned U.S. Pat. No. 6,352,991; Qiu, Y. L., et al., J. Med.Chem. 41:10–23 (1998); Antiviral Chem. Chemother. 9:341–352 (1998)).However, there continues to be a need for novel compounds which areactive against pathogenic viruses, including HCMV, HSV-1, HSV-2, HHV-6,HIV, and hepatitis B virus (HBV).

SUMMARY OF THE INVENTION

The present invention provides novel2-amino-9-[(2-hydroxymethyl)cyclo-propylidenemethyl]purines, includingall geometric and optically active isomers, prodrugs andpharmacologically acceptable salts thereof. These compounds have beenfound to be useful antiviral agents and are effective against HCMV,HSV-1, HSV-2, HHV-6, HIV, EBV and HBV, as well as against otherpathogenic viruses.

The compounds of the present invention have the following Formulas:

-   wherein B is 2-aminopurine-9-yl, which may be unsubstituted or    substituted in the 6 position with NHR₁, OR₂, or SR₃;-   R₁ is selected from the group consisting of alkyl, alkenyl, alkynyl,    and C₄₋₁₈ cycloalkyl, any of which may be optionally substituted    with one or more members of the group consisting of hydroxy, halo,    amino, acyl, cycloalkyl, heterocyclyl and aryl;-   R₂ is selected from the group consisting of C₂₋₁₈ alkyl, alkenyl,    alkynyl, and cycloalkyl, any of which may be branched or unbranched    and optionally substituted with one or more members of the group    consisting of hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl,    and aryl; and-   R₃ is selected from the group consisting of alkyl, alkenyl, alkynyl,    and cycloalkyl, any of which may be branched or unbranched and    optionally substituted with one or more members of the group    consisting of hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl    and aryl.

The compounds of the present invention also include, including allgeometric and optically active isomers, prodrugs and pharmacologicallyacceptable salts of the novel2-amino-9-[(2-hydroxymethyl)cyclopropylidene-methyl]purine compounds ofthis invention.

Compositions of this invention useful for treating viral infections,such as HCMV, HSV-1, HSV-2, HHV-6, HIV, EBV and HBV, comprise aneffective amount of at least one compound according to this invention ora pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.

Additional objects, advantages, and features of the present inventionwill become apparent from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Schemes 1 and 2 are illustrative syntheses of some compounds of thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the following terms shall be defined as follows (unlessotherwise noted):

“Acyl” shall mean the radical C(O)R, wherein R is selected from alkyl,aryl, alkylaryl, arylakyl (such as benzyl), alkylarylalkyl,heterocyclyl, heterocyclylalkyl, carbocyclyl, carbocyclylalkyl,alkoxyalkyl (such as methoxymethyl), alkoxyalkyl, aryloxyalkyl (such asphenoxymethyl), poly(alkyloxy)alkyl (such as polyethers like poly(methoxy)methyl), aryl (such as phenyl optionally substituted with halo,lower alkyl or lower alkoxy), arylalky, and alkylaryl. Specific examplesof acyl moieties include, without limitation, acetyl, propionyl,butyryl, pentanoyl, 3-methylbutyryl, hydrogen succinate,3-chlorobenzoate, benzoyl, acetyl, pivaloyl, mesylate, propionyl,valeryl, caproic, caprylic, capric, lauric, myristic, palmitic, stearicand oleaoic.

“Alkyl” shall mean a saturated straight chain or branched, primary,secondary, or tertiary hydrocarbon radical that is fully saturated,typically C₁–C₁₈, preferably C₁–C₁₀, and more preferably C₁–C₆ Preferredalkyl groups include, without limitation, methyl, ethyl, propyl, butyl,pentyl, hexyl, isopropyl, isobutyl, sec-butyl, t-butyl, isopentyl, amyl,and t-pentyl. In the case of R₂, preferred alkyl groups are C₂–C₁₈,preferably C₂–C₁₀, and more preferably C₂–C₆, and include, withoutlimitation, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl,sec-butyl, t-butyl, isopentyl, amyl, and t-pentyl.

“Alkenyl” shall mean an alkyl moiety having at least one double bond.Alkenyl groups containing three or more carbon atoms may be straight orbranched.

“Alkynyl” shall mean an alkyl moiety having at least one triple bond.Alkynyl groups containing three or more carbon atoms may be straight orbranched.

“Amino acid” shall mean an amino acid or amino acid residue selectedfrom the group of all of the naturally occurring amino acids, thoseamino acids in their D- and L-configurations, and the known non-native,synthetic, and modified amino acids, such as homocysteine, ornithine,norleucine and beta.-valine. A list of non natural amino acids may befound in “The Peptides”, vol 5, 1983, Academic Press, Chapter 6 by D. C.Roberts and F. Vellaccio. In the context of the substituents R₁X or R₂Xin Formulas 3 and 4, preferred amino acid residues include alanine andtryptophan.

“Aryl” shall mean a mono- or polycyclic aromatic ring or ring systemwhich may be carbocyclic or heterocyclic and may be unsubstituted orsubstituted with one or more substituents selected from (but not limitedto) alkyl (preferably, lower alkyl), hydroxy, alkoxy (preferably, loweralkoxy), alkylthio, cyano, halo, amino, and nitro). Examples of arylgroups are phenyl, methylphenyl, dimethylphenyl, aminophenyl,nitrophenyl, hydroxyphenyl, and the like.

“Cycloalkyl” shall mean a mono-, bi- or polycyclic alkyl radical. Forconvenience, the term “cycloalkyl” shall also expressly includecycloalkenyl cycloalkynyl radicals. A “branched cycloalkyl” shall mean acycloalkyl ring in which one or more ring members are substituted withalkyl. In general, these rings shall typically be C₃–C₁₈, preferablyC₃–C₁₀, and more preferably C₃–C₈. In the case of R₁, the cycloalkylradical is preferably C₄–C₁₈, more preferably, C₄–C₁₂ and mostpreferably, C₄–C₁₀.

“Halo” shall mean fluoro, chloro, bromo, or iodo.

“Heterocyclyl” shall mean a mono-, bi- or polycyclic radical containingone or more rings which may be saturated, unsaturated, or aromatic,wherein at least one ring contains one or more heteroatoms selected fromnitrogen (N), oxygen (O), and sulfur (S). Heterocyclyl radicalstypically have 3–18 total ring members and preferably 3–10 total ringmembers Preferably, heterocyclyl radicals are monocyclic (preferablyhaving 3–8 and more preferably, 3–6 ring members) or bicyclic(preferably having 6–12 ring members and more preferably, 8–10 ringmembers). Suitable heterocyclyl for use in the compounds of thisinvention include radicals of (without limitation) furan, dioxolane,thiophene, pyrrole, pyrazole, triazole, imidazole, pyrrolidine, pyran,pyridine, pyrimidine, morpholine, piperidine, oxazole, isoxazole,oxazoline, oxazolidine, oxathiazole, thiazole, isothiazole, thiadiazole,tetrazole, benzofuran, indole, isoindole, quinazoline, quinoline,isoquinoline, purine, pyrrolopyrimidine, pyrrazolopyrimidine, pteridine,ketal. In addition, heterocyclyl radicals may contain one or moresubstituents (i.e., a ring substituent, such as a halogen atom, an alkylradical, or aryl radical) attached to a ring member atom of theheterocyclyl radical. All stable isomers of heterocyclyl groups arecontemplated in this definition.

“Lower” shall mean the group to which it is applied preferably has 1–6,and more preferably 1–4, carbon atoms, except in the case of rings (suchas cycloalkyl and heterocyclyl), in which case “lower” signifies 3–6ring members.

“Patient” shall mean any warm-blooded mammal, including withoutlimitation, a human.

“Pharmaceutically acceptable salts” shall mean those salts of anycompound of this invention derived from an inorganic or organic acid orbase recognized in the art as compatible for pharmaceuticalcompositions. Examples of suitable acids include hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, formic, benzoic, malonic,naphthalene-2-sulfonic and benzenesulfonic acids. Other acids such asoxalic, while not in themselves pharmaceutically acceptable, may beuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts. Salts derivedfrom appropriate bases include alkali metal (e.g., sodium, potassium),alkaline earth metal (e.g., magnesium), ammonium and NR₄ ⁺ (where R is aC₁₋₄ alkyl) salts, and the like. Reference to a compound according tothe invention is understood to include any and all correspondingpharmaceutically acceptable salts thereof. For convenience, the terms“pharmaceutical” and “pharmaceutically acceptable” are understood toencompass compounds acceptable for the practice of veterinary medicineas well.

“Pharmaceutically acceptable carriers” for use in the formulations ofthis invention are carriers that are compatible with the otheringredients of the formulation and not deleterious to the recipientthereof.

“Therapy” and “therapeutic” shall mean treatment of an individual for aviral infection or disease. For convenience, these terms are alsounderstood to encompass prophylactic or precautionary use oradministration of a compound of this invention. Such precautionary orprophylactic use is exemplified by administration of an antiviral agentto an individual(s) suspected, but not proven, of having a viralinfection or to an individual(s) susceptible to contracting a pathogenicviral infection due to contact with contaminated items, or contact withother individuals carrying a contagious viral disease.

All published documents referred to herein are expressly incorporatedherein by reference.

The compounds of the present invention which have been found to beeffective against pathogenic viruses including HCMV, HSV-1, HSV-2,HHV-6, HIV, EBV and HBV, are compounds of Formulas 1 and 2:

-   wherein B is 2-aminopurine-9-yl, which may be unsubstituted or    substituted in the 6 position with NHR₁, OR₂, or SR₃;-   R₁ is selected from the group consisting of alkyl, alkenyl, alkynyl,    and C₄₋₁₈ cycloalkyl, any of which may be optionally substituted    with one or more members of the group consisting of hydroxy, halo,    amino, acyl, cycloalkyl, heterocyclyl and aryl;-   R₂ is selected from the group consisting of C₂₋₁₈ alkyl, alkenyl,    alkynyl, and cycloalkyl, any of which may be branched or unbranched    and optionally substituted with one or more members of the group    consisting of hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl,    and aryl; and-   R₃ is selected from the group consisting of alkyl, alkenyl, alkynyl,    and cycloalkyl, any of which may be branched or unbranched and    optionally substituted with one or more members of the group    consisting of hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl    and aryl.

In a preferred embodiment, B is 2-aminopurine-9-yl, which is substitutedin the 6 position with NHR₁, OR₂, or SR₃; wherein

-   R₁ is selected from the group consisting of C₁–C₈ alkyl, C₃–C₈    alkenyl, C₃–C₈ alkynyl, and C₄₋₁₀ cycloalkyl, any of which may be    optionally substituted with one or more members of the group    consisting of hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl    and aryl;-   R₂ is selected from the group consisting of C₂₋₈ alkyl, C₃–C₈    alkenyl, C₃–C₈ alkynyl, and C₃₋₁₀ cycloalkyl, any of which may be    branched or unbranched and optionally substituted with one or more    members of the group consisting of hydroxy, halo, amino, acyl,    cycloalkyl, heterocyclyl, and aryl; and-   R₃ is selected from the group consisting of C₂₋₈ alkyl, C₃–C₈    alkenyl, C₃–C₈ alkynyl, and C₃₋₁₀ cycloalkyl, any of which may be    branched or unbranched and optionally substituted with one or more    members of the group consisting of hydroxy, halo, amino, acyl,    cycloalkyl, heterocyclyl and aryl.

In another preferred embodiment, B is 2-aminopurine-9-yl, which issubstituted in the 6 position with NHR₁, OR₂, or SR₃; wherein

-   R₁ is selected from the group consisting of C₁–C₆ alkyl, C₃–C₆    alkenyl, C₃–C₆ alkynyl, and C₄₋₈ cycloalkyl, any of which may be    optionally substituted with one or more members of the group    consisting cycloalkyl, heterocyclyl and aryl;-   R₂ is selected from the group consisting of C₂₋₆ alkyl, C₃–C₆    alkenyl, C₃–C₆ alkynyl, and C₃₋₈ cycloalkyl, any of which may be    branched or unbranched and optionally substituted with one or more    members of the group consisting of cycloalkyl, heterocyclyl, and    aryl; and-   R₃ is selected from the group consisting of C₂₋₆ alkyl, C₃–C₆    alkenyl, C₃–C₆ alkynyl, and C₃₋₈ cycloalkyl, any of which may be    branched or unbranched and optionally substituted with one or more    members of the group consisting of hydroxy, halo, amino, acyl,    cycloalkyl, heterocyclyl and aryl.

Preferred compounds of the present invention are:

-   (Z,S)-(+)-2-Amino-6-allylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-propargylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-cyclopropylmethylamino-9-[(2-hydroxymethyl)-cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-isopropylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-benzylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-cyclohexylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-(2-hydroxy)ethylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-propoxy-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-pentyloxy-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-allyloxy-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-cyclopropylmethoxy-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-propylthio-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-pentylthio-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;-   (Z,S)-(+)-2-Amino-6-thio-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;    and-   (Z,S)-(+)-2-Amino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine.

Prodrugs of the antiviral compounds of the present invention includelipophilic phosphate esters or amidates capable of penetrating the cellmembrane. Those skilled in the art will appreciate that the aim ofprodrugs is to provide effective therapeutic agents for resistantstrains of viruses (McGuigan, C., et al., J. Med. Chem. 36:1048–1052(1993)) or activate inactive analogs (Franchetti, P., et al., J. Med.Chem. 37:3534–3541 (1994)). Preferred prodrugs include the phosphateesters of Formulas 3 and 4:

-   wherein-   B is defined as above for Formulas 1 and 2;-   X is O; and-   R₁ and R₂ are alkyl or aryl. The R₁X or R₂X may also be amino acid    residues (such as alanine) with X as NH.

The nomenclature of the compounds of the present invention followsstandard conventions. For the sake of clarity, the purine ring B isnumbered according to the standard convention as indicated below:

It is appreciated that heterocyclic rings containing hydroxy and aminogroups are tautomeric with the corresponding oxo and imino compounds.

The compounds described by Formulas 1 through 4 contain an asymmetriccarbon atom. Compounds of Formula 1 and 2 of the present invention aretherefore racemic mixtures of two optical antipodes which may beresolved by conventional methods such as chromatography or fractionalcrystallization of suitable diastereoisomeric derivatives such as saltsor esters with optically active acids (camphor 10-sulfonic acid,methoxyacetic acid, dibenzoyltartaric acid,6-methoxy-2-naphthyl-2-propanoic acid, etc.), by an enantioselectiveenzymic synthesis of esters of one antipode such as acetates orbutyrates or by an enantioselective enzymic hydrolysis of esters ofcompounds of Formulas 1 and 2, such as acetates or butyrates. Thesuitable enzymes include, but are not limited to, lipases such as lipaseAK, lipase P30 or esterases such as pig liver esterase. Racemiccompounds containing adenine moiety may also be resolved by the actionof adenosine deaminase. Alternatively, the R- and S-enantiomers can beobtained by synthetic methods utilizing enantiomerically pure startingmaterials. The compounds of this invention expressly include the racemicmixture and the individual optically isomers (e.g., R- andS-enantiomers) of each of these compounds, their pharmaceuticallyacceptable salts and prodrugs thereof. Preferred compounds of thepresent invention include the individual R- and S-enantiomers of thecompounds of Formulas 1–4 and more preferably, the R- and S-enantiomersof the compounds of Formulas 1 and 2.

Compounds of formulas 3 and 4 derived from racemic analogues ofcompounds of Formulas 1 and 2 will be mixtures of four diastereoisomers(provided that R₁X is not the same as R₂X).

In Schemes 1 and 2 attached hereto,(Z)-2-amino-6-chloro-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(Compound 5, see Scheme 1) is used as a convenient starting material forcompounds of this invention. This compound can be obtained in racemic orenatiomeric (R or S) form along with the corresponding E-isomer (Qiu etal., J. Med. Chem. 1998, 41, 10–23; Qiu and Zemlicka, Synthesis 199811447–1452; Qiu et al., Antiviral Chem. Chemother. 2000, 11 191–202;Chen and Zemlicka, J. Org. Chem. 2002, 67, 286–289). For purpose ofillustration, the S-enantiomer is shown in Compound 5). The reactivityof the chlorine atom of Compound 5 provides for easy substitution withamines, as shown, for example, in Scheme 1 (Compounds 6–12). Suchsubstitution may be conducted (for example) with alcohols (Scheme 2,Compounds 13a–15), thiols (Scheme 2, Compounds 16a and 16b), and sodiumhydroxulfide (Scheme 2, Compound 17). Reduction of Compound 17 gives the6-unsubstituted 2-aminopurine derivative, Compound 18. These procedurescan be readily modified by those of ordinary skill in the art to produceother examples of compounds of this invention. For example, thisprocedure may be used with the E-isomer of Compound 5.

Compositions within the scope of invention include those comprising anovel compound of the present invention in an effective amount toachieve an intended purpose, such as antiviral efficacy. Determinationof an effective amount and intended purpose is within the skill of theart. Preferred dosages, which are dependent for example, on the severityof the infection and the individual patient's response to the treatment,can range from about 0.01 to about 100 mg/kg of body weight per day(preferably in the range of 0.5 to 60 mg/kg/day, most preferably in therange of 1 to 20 mg/kg/day) to give a blood concentration ranging fromabout 0.05 Φg to about 5 mg per mL of whole blood (preferably, 1 toabout 75 μM, more preferably about 2 to 50 μM, most preferably about 3to about 30 μM).

Pharmaceutical formulations include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), transdermal, vaginalor parenteral (including intramuscular, sub-cutaneous and intravenous)administration or in a form suitable for administration by inhalation orinsufflation. The formulations may, where appropriate, be convenientlypresented in discrete dosage units and may be prepared by any of themethods well known in the art of pharmacy. All methods include the stepof bringing into association the active compound with liquid carriers orfinely divided solid carriers or both and then, if necessary, shapingthe product into the desired formulation.

Pharmaceutically acceptable compositions of the present invention mayinclude suitable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichmay be used pharmaceutically. Such preparations, preferably those whichcan be administered orally, include tablets, dragees, liquids, caplets,and capsules. Further preferred preparations are those which can beadministered vaginally or rectally, such as suppositories, as well assuitable solutions for administration by injection or orally, containfrom about 0.1 to about 99%, preferably about 25 to about 85%, of theactive compound of the present invention, together with the excipient.

The pharmaceutical compositions of the present invention aremanufactured in a manner which is itself known, e.g., using theconventional mixing, granulating, dragee-making, dissolving orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding a resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as sugars, e.g.,lactose or sucrose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, e.g., tricalcium diphosphate or calcium hydrogenphosphate, as well as binders such as starch paste, using, e.g., maizestarch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose and/or polyvinylpyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, e.g., silica,talc, stearic acid or salts thereof, such as magnesium stearate orcalcium stearate, and/or polyethylene glycol. Dragee cores are providedwith suitable coatings which, if desired, are resistant to gastricjuices. For this purpose, concentrated sugar solutions may be used,which may optionally contain gum arabic, talc, polyvinylpyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvent or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations, such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate, are used. Dyestuffs or pigmentsmay be added to the tablets or dragee coatings, e.g., for identificationor in order to characterize different combinations of active compounddoses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules may contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycols. In addition, stabilizers maybe used.

Possible pharmaceutical preparations which can be used rectally include,e.g., suppositories, which consist of a combination of the activecompounds with a suppository base. Suitable suppository bases are, e.g.,natural or synthetic triglycerides, paraffin hydrocarbons, polyethyleneglycols or higher alkanols. It is also possible to use gelatin rectalcapsules which consist of a combination of the active compounds with abase. Possible base materials include, e.g., liquid triglycerides,polyethylene glycols or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, e.g.,water-soluble salts. In addition, suspension of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, such as sesame oil,or synthetic fatty acid esters, e.g., ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension such as sodium carboxymethylcellulose,sorbitol and/or dextran. Optionally, the suspension may also containstabilizers.

When desired, the above described formulations may be adapted to give asustained or time-delayed release of compound of the invention using anyof the sustained or time-delayed formats available in art.

Alternatively, the active compounds of the present invention may beadministered in the form of liposomes, pharmaceutical compositionswherein the active compound is contained either dispersed or variouslypresent in corpuscles consisting of aqueous concentrate layers adherentto hydrophobic lipidic layer. The active compound may be present both inthe aqueous layer and in the lipidic layer or in the non-homogeneoussystem generally known as a lipophilic suspension.

It will be appreciated that the active compounds of the presentinvention may be administered in combination with other therapeuticagents, including known antiviral agents, e.g., acyclovir, ganciclovir,zidovudine, AZT, ddI, ddC, 3TC and d4T. The individual components ofsuch combinations may be administered either sequentially orsimultaneously in separate or combined pharmaceutical formulations. Whena compound of the invention or a pharmaceutically acceptable saltthereof is used in combination with a second therapeutic compound, thedose of each compound may be either the same as or differ from that whenthe compound is used alone. Appropriate doses will be readilyappreciated by those skilled in the art.

The ratio between a compound of the present invention and a secondtherapeutic agent will be readily appreciated by those skilled in theart. For example, one may use a ratio in the range from about 1:1 toabout 1:50 (by weight) of compound of the invention:second therapeuticagent. In a further embodiment, one may use a ratio ranging from about1:1 to about 1:30 (by weight) of a compound of the invention:secondtherapeutic agent. In a further embodiment, one may use a ratio fromabout 1:1 to about 1:20 (by weight) of a compound of theinvention:second therapeutic agent. In a further embodiment, one may usea ratio in the range from about 1:1 to about 1:15 (by weight) of acompound of the invention:second therapeutic agent. In a furtherembodiment, one may use a ratio ranging from about 1:1 to about 1:10 (byweight) of a compound of the invention:second therapeutic agent. In afurther embodiment, one may use a ratio in the the range from about 1:1to about 1:5 (by weight) of a compound of the invention:secondtherapeutic agent. In yet a further embodiment, one may use a ratio inthe range of about 1:1 to about 1:3 (by weight) of a compound of theinvention:second therapeutic agent. If more than one additionaltherapeutic agent is used in combination with a compound of thisinvention, the above ratios may be adjusted accordingly.

EXAMPLES

The following Examples further describe the compounds of the presentinvention and the synthesis schemes for producing same (reference shouldbe made to Schemes 1 and 2 above for compound numbers). These Examplesare provided to illustrate various embodiments of the present inventionand shall not be considered as limiting in scope.

Compound 5 was prepared as described (Chen, X.; Zemlicka, J. J. Org.Chem. 2002, 67, 286–289).

Example 1(Z,S)-(+)-2-Amino-6-allylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(6)

A mixture of compound 5 (503 mg, 2.0 mmol) and allylamine (680 mg, 12mmol) in ethanol (20 mL) was stirred overnight at room temperature.Volatile components were evaporated in vacuo and the residue waschromatographed on a silica gel column using dichloromethane-methanol(95:5) to give product 6 (445 mg, 82%).

Mp. 168–170° C. [α]²⁰ _(D) 70.2° (c=0.45, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 500 MHz) δ1.14–1.17 (m, 1H), 1.42–1.46 (m, 1H), 2.06–2.09 (m,1H),3.30–3.35 (m, 1H), 3.66–3.70 (m, 1H). 4.04 (bs, 2H), 5.00–5.14 (m,1H), 5.88–5.95 (m, 1H), 5.97 (bs, 2H), 7.17 (d, 1H, J=1.5 Hz), 7.48 (bs,1H), 8.33 (s, 1H). ¹³C NMR (DMSO-d₆, 125 MHz) ppm 6.9, 19.8, 42.4, 63.7,111.0, 114.2, 115.4, 134.7, 136.8, 150.4, 155.4, 161.2. ESI-MS(NaCl+KCl) 567 (2M+Na, 60), 311 (M+K, 15), 295 (M+Na, 58), 273 (M+H,100). Calculated for C₁₃H₁₆N₆O: C, 57.34; H, 5.92; N, 30.86. Found: C,57.32; H, 5.82; N, 30.91.

Example 2(Z,S)-(+)-2-Amino-6-propargylamino-9-[(2-hydroxymethyl)-cyclopropylidenemethyl]purine(7)

A mixture of compound 5 (190 mg, 0.75 mmol) and propargylamine (270 mg,4.8 mmol) in ethanol (18 mL) was stirred at 50° C. for 1 h and thestirring was continued for 16 h at room temperature. The work-upfollowed the procedure described in Example 1 to give compound 7 (175mg, 85%).

Mp. 192–194° C. [α]²⁰ _(D) 77.3° (c=0.40, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 500 MHz) δ1.15–1.18 (m, 1H), 1.43–1.47 (m, 1H), 2.03–2.14 (m,1H), 3.03 (s, 1H), 3.29–3.35 (m, 1H), 3.67 (m, 1H), 4.19 (d, 2H, J=3.2Hz), 5.07 (t, 1H, J=5.0 Hz), 6.08 (bs, 2H), 7.18 (d, 1H, J=2.5 Hz), 7.69(bs, 1H), 8.36 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) ppm 6.9, 19.8, 29.3,63.7, 73.1, 83.0, 110.9, 113.6, 114.4, 135.1, 154.8, 161.1. ESI-MS(NaCl) 563 (2M+Na, 80), 293 (M+Na, 100.0), 271 (M+H, 18). Calculated forC₁₃H₁₄N₆O: C, 57.77; H, 5.22; N, 31.09. Found: C, 57.81; H, 5.39; N,31.11.

Example 3(Z,S)-(+)-2-Amino-6-cyclopropylmethylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(8)

A mixture of compound 5 (200 mg, 0.8 mmol) and cyclopropylmethylamine(360 mg, 5.0 mmol) in ethanol (15 mL) was stirred at 50° C. for 16 h.The work-up followed the procedure described in Example 1 to givecompound 8 (201 mg, 88%).

Mp. 180–182° C. [α]²⁰ _(D) 68.4° (c=0.28, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 400 MHz) δ1.06–1.12 (m, 1H), 1.14–1.17 (m, 1H), 1.44 (dt, 1H,J=8.4 Hz and 1.6 Hz), 2.04–2.09 (m, 1H), 3.26–3.36 (m, 3H), 3.66–3.72(m, 1H), 5.10 (t, 1H, J=5.2 Hz), 5.97 (bs, 2H), 7.19 (s, 1H), 7.35 (bs,1H), 8.33 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) ppm 4.0, 6.9, 12.0, 19.9,44.5, 63.7, 111.1, 113.6, 114.1, 134.6, 150.3, 155.5, 161.2. ESI-MS(NaCl) 309 (M+Na, 55), 287 (M+H, 100). Calculated for C₁₄H₁₈N₆O: C:58.73, H, 6.34; N, 29.35. Found: C, 58.95; H, 6.33; N, 29.43.

Example 4(Z,S)-(+)-2-Amino-6-isopropylamino-9-[(2-hydroxymethyl)-cyclopropylidenemethyl]purine(9)

A mixture of compound 5 (250 mg, 1.0 mmol) and isopropylamine (590 mg,10 mmol) in ethanol (10 mL) was stirred at room temperature for 16 h.The work-up followed the procedure described in Example 1 to givecompound 9 (215 mg, 80%).

Mp. 153–155° C. [α]²⁰ _(D) 67.8° (c=0.36, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 500 MHz) δ1.15 (d, 6H, J=6.5 Hz), 1.14–1.16 (m, 1H), 1.43 (m,1H), 2.03–2.12 (m, 1H), 3.30 –3.35 (m, 1H), 3.67–3.69 (m, 1H), 4.37 (m,1H), 5.10 (t, 1H, J=5.0 Hz), 5.94 (bs, 2H), 7.01 (bs, 1H), 7.18 (s, 1H),8.32 (s, 1H). ¹³C NMR(DMSO-d₆, 125 MHz) ppm 6.9, 19.8, 23.3, 41.4, 63.7,111.1, 113.5, 114.1, 134.5, 150.3, 154.9, 161.2. ESI-MS(NaCl)571(2M+Na,50),297 (M+Na, 50), 275 (M+H, 100). Calculated for C₁₃H₁₈N₆0: C,56.92; H, 6.61; N, 30.64. Found: C, 57.09; H, 6.40; N, 30.76.

Example 5(Z,S)-(+)-2-Amino-6-benzylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(10)

A mixture of compound 5 (250 mg, 1.0 mmol) and benzylamine (645 mg, 6.0mmol) in ethanol (15 mL) was stirred at 50° C. for 16 h. The work-upfollowed the procedure described in Example 1 to give afterchromatography in dichloromethane-methanol (97:3) compound 10 (240 mg,75%).

Mp. 133–135° C. [α]²⁰ _(D) 69.70 (c=0.35, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 400 MHz) δ1.14–1.18 (m, 1H), 1.43–1.48 (m, 1H), 2.03–2.11 (m,1H), 3.28–3.32 (m, 1H), 3.65–3.70 (m, 1H), 4.62 (s, 2H), 5.07 (t, 1H,J=5.0 Hz), 5.96 (bs, 2H), 7.17–7.20 (m, 2H), 7.25–7.30 (m, 4H), 7.86(bs, 1H), 8.32 (s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) ppm 6.9, 19.9, 43.2,63.7, 111.1, 113.6, 114.3, 127.2, 127.9, 128.8, 134.8, 141.2, 150.5,155.5, 161.2. ESI-MS (NaCl) 345 (M+Na, 60), 323 (M+H, 100). Calculatedfor C₁₇H₁₈N₆O: C. 63.34; H, 5.63; N, 26.07. Found: C, 63.57; H, 5.67; N,25.93.

Example 6(Z,S)-(+)-2-Amino-6-cyclohexylamino-9-[(2-hydroxymethyl)-cyclopropylidenemethyl]purine(11)

A mixture of compound 5 (200 mg, 0.8 mmol) and cyclohexylamine (645 mg,6.5 mmol) in ethanol (15 mL) was refluxed with stirring for 16 h. Thework-up followed the procedure described in Example 5 to give compoundII (205 mg, 82%).

Mp. 183–185° C. [α]²⁰ _(D) 81.2° (c=0.34, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 400 MHz) δ1.07–1.29 (m, 6H), 1.44 (t, 1H, J=8.0 Hz), 1.56 (d,1H, J=11.6 Hz), 1.67–1.70 (m, 2H), 1.78–1.88 (m, 2H), 2.00–2.11 (m, 1H),3.30–3.35 (m, 1H), 3.66–3.69 (m, 1H), 4.01 (bs, 1H), 5.10 (t, 1H, J=5.2Hz), 5.93 (bs, 2H), 6.95 (bs, 1H), 7.18 (s, 1H), 8.31 (s, 1H). ¹³C NMR(DMSO-d₆, 100 MHz) ppm 6.9, 19.9, 25.8, 25.9, 33.3, 48.6, 63.7, 111.1,113.4, 114.1, 134.5, 150.5, 154.8, 161.2. ESI-MS (NaCl) 337 (M+Na, 30),315 (M+H, 100). Calculated for C₁₆H₂₂N₆O: C, 61.13; H, 7.05; N, 26.73.Found: C, 61.32; H, 6.95; N, 26.92.

Example 7(Z,S)-(+)-2-Amino-6-(2-hydroxy)ethylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(12)

A mixture of compound 5 (200 mg, 0.80 mmol) and (2-hydroxy)-ethylamine(250 mg, 4.1 mmol) in ethanol (15 mL) was stirred at 50° C. for 16 h.The work-up followed the procedure described in Example 1 to give afterchromatography using dichloromethane-methanol (9:1) compound 12 (171 mg,77%).

Mp. 150–153° C. [α]²⁰ _(D) 76.8° (c=0.32, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 400 MHz) δ1.14–1.18 (m, 1H), 1.42–1.46 (m, 1H), 2.05–2.09 (m,1H), 3.30–3.53 (m, 5H), 3.68 (dd, 1H, J=10.4 Hz and 5.6 Hz), 4.82 (bs,1H), 5.09 (bs, 1H), 5.98 .(bs, 2H), 7.13 (bs, 1H), 7.18 (s, 1H), 8.32(s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) ppm 6.9, 19.8, 43.0, 60.8, 63.7,111.1, 113.7, 114.3, 134.8, 150.3, 155.7, 161.2. ESI-MS (NaCl+KCl) 315(M+K, 10), 299 (M+Na, 100), 277 (M+Na, 95). Calculated for C₁₇H₁₈N₆O: C,52.17; H, 5.84; N, 30.42. Found: C, 52.36; H, 5.92; N, 30.56.

Example 8(Z,S)-(+)-2-Amino-6-propoxy-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(13a)

A mixture of compound 5 (200 mg, 0.8 mmol), potassium carbonate (166 mg,12 mmol), and 1-propanol (6 mL) was stirred at 70–80° C. for 16 h. Thework-up followed the procedure described in Example 1 to give compound13a (188 mg, 85%).

Mp. 147–149° C. [α]²⁰ _(D) 68.3° (c=0.42, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 500 MHz) δ0.94 (t, 3H, J=7.2 Hz), 1.15–1.18 (m, 1H), 1.43–1.46(m, 1H), 1.71–1.78 (m, 2H), 2.06–2.12 (m, 1H), 3.27–3.32 (m, 1H),3.69–3.73 (m, 1H), 4.34 (t, 2H, J=6.8 Hz), 5.08 (t, 1H, J=5.0 Hz), 6.49(s, 2H), 7.20 (d, 1H, J=1.5 Hz), 8.47 (s, 1H). 13C NMR (DMSO-d₆, 125MHz) ppm 6.9, 10.9, 19.9, 22.5, 63.6, 68.0, 110.8, 114.1, 115.2, 137.1,153.3, 160.8, 161.2. ESI-MS (KCl) 573 (2 M+Na, 100), 314 (M+K, 10), 298(M+Na, 50), 276 (M+H, 10). Calculated for C₁₃H₁₇N₅O₂: C, 56.71; H, 6.22;N, 25.44. Found: C, 56.91; H, 6.39; N, 25.18.

Example 9(Z,S)-(+)-2-Amino-6-pentyloxy-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(13b)

A mixture of compound 5 (150 mg, 0.6 mmol), potassium carbonate (210 mg,1.5 mmol), 1-pentanol (0.4 mL, 3.7 mmol) and 1,2-dimethoxyethane (10 mL)was stirred at 80° C. for 16 h. The work-up followed the proceduredescribed in Example 1. Chromatography using dichloromethane-methanol(96:4) afforded compound 13b (155 mg, 85%).

Mp. 135–137° C. [α]²⁰ _(D) 77.7° (c=0.26, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 400 MHz) δ0.87 (t, 3H, J=6.0 Hz), 1.16–1.19 (m, 1H), 1.25–1.40(m, 4H), 1.42–1.49 (m, 1H), 1.67–1.78 (m, 2H), 2.05–2.14 (m, 1H),3.27–3.35 (m, 1H), 3.68–3.73 (m, 1H), 4.38 (t, 2H, J=6.6 Hz), 5.07 (t,1H, J=5.4 Hz), 6.49 (bs, 2H), 7.20 (s, 1H), 8.47 (s, 1H). ¹³C NMR(DMSO-d₆, 125 MHz) ppm 6.9, 14.6, 19.9, 22.6, 28.3, 28.8, 63.6, 66.4,110.8, 114.1, 115.2, 137.0, 153.3, 160.8, 161.2. ESI-MS (NaCl+KCl) 645(2M+K, 5), 629 (2 M+Na, 30), 607 (2M+H, 85), 304 (M+H, 100). Calculatedfor C₁₅H₂₁N₅O₂: C, 59.39; H, 6.98; N, 23.09. Found: C, 59.49; H, 7.02;N, 23.12.

Example 10(Z,S)-(+)-2-Amino-6-allyloxy-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(14)

A mixture of compound 5 (200 mg, 0.8 mmol), potassium carbonate (165 mg,12 mmol) and allyl alcohol (5 mL) was stirred at 60–70° C. for 16 h. Thework-up followed the procedure described in Example 1 to give compound14 (172 mg, 79%).

Mp. 163–165° C. [α]²⁰ _(D) 68.40 (c=0.23, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 300 MHz) δ1.15–1.20 (m, 1H), 1.43–1.49 (m, 1H), 2.07–2.02 (m,1H), 3.26 (m, 1H), 3.68–3.75 (m, 1H), 4.94 (d, 2H, J=6.0 Hz), 5.07 (t,1H, J=5.2 Hz), 5.25 (dd, 1H, J=7.5 and 1.8 Hz), 5.40 (dd, 1H, J=17.1 Hz,1.8 Hz), 6.03–6.16 (m, 1H), 6.53 (s, 2H), 7.20 (d, 1H, J=1.2 Hz), 8.49(s, 1H). ¹³C NMR (DMSO-d₆, 75 MHz) ppm 6.9, 19.9, 63.6, 66.8, 110.8,114.0, 115.4, 118.8, 133.9, 137.3, 153.4, 160.6, 160.7. ESI-MS (NaCl)569 (2M+Na, 100), 296 (M+Na, 48), 312 (M+K, 10), 274 (M+H, 10).Calculated for C₁₃H₁₅N₅O₂: C, 57.13; H, 5.53; N, 25.63. Found: C, 57.43;H, 5.44; N, 25.52.

Example 11(Z,S)-(+)-2-Amino-6-cyclopropylmethoxy-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(15)

A mixture of compound 5 (150 mg, 0.6 mmol), potassium carbonate (210 mg,1.5 mmol) and cyclopropylmethanol (2 mL) in 1,2-dimethoxyethane (7 mL)was stirred at 80° C. for 16 h. The workup followed the proceduredescribed in Example 9 to give compound 15 (142 mg, 82%).

Mp. 129–130° C. [α]²⁰ _(D) 74.5° (c=0.33, N,N-dimethylfornamide). ¹H NMR(DMSO-d₆, 400 MHz) δ0.34 (d, 2H, J=4.0 Hz), 0.57 (d, 2H, J=7.2 Hz),1.16–1.20 (m, 1H), 1.25–1.33 (m, 1H), 1.47 (t, 1H, J=8.4 Hz), 2.05–2.14(m, 1H), 3.27–3.29 (m, 1H), 3.69–3.73 (m, 1H), 4.23 (d, 2H, J=7.2 Hz),5.06 (t, 1H, J=5.4 Hz), 6.49 (bs, 2H). 7.21 (s, 1H), 8.48 (s, 1H). ¹³CNMR (DMSO-d₆, 100 MHz) ppm 4.0, 6.9, 10.7, 19.9, 63.6, 71.1, 110.8,114.1, 115.3, 137.1, 153.3, 160.8, 161.1. ESI-MS (NaCl+KCl) 613 (M+K,5), 597 (2M+Na), 15), 575 (2M+1, 28), 288 (M+H, 100). Calculated forC₁₄H₁₇N₅O₂: C, 58.52; H, 5.96; N, 24.37. Found: C, 58.49; H, 6.03; N,24.29.

Example 12(Z,S)-(+)-2-Amino-6-propylthio-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(16a)

The mixture of compound 5 (125 mg, 0.5 mmol), potassium carbonate (140mg, 1.0 mmol), 1-propanethiol (750 mg, 10 mmol) and 1,2-dimethoxyethane(6 mL) was stirred at 50° C. for 10 h. The work-up followed theprocedure described in Example 1 to give compound 16a (105 mg, 72%).

Mp. 163–164° C. [α]²⁰ _(D) 77.3° (c=0.39, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 500 MHz) δ0.97 (t, 3H, J=7.5 Hz), 1.16–1.19 (m, 1H), 1.44–1.47(m, 1H), 1.62–1.69 (m, 2H), 2.08–2.11 (m, 1H), 3.23 (t, 2H, J=7.0 Hz),3.27–3.31 (m, 1H), 3.70–3.71 (m, 1H), 5.05 (t, 1H, J=4.0 Hz), 6.57 (s,2H), 7.19 (s, 1H), 8.53 (s, 1H). ¹³C NMR (DMSO-d₆, 125 MHz) ppm 6.9,13.9, 19.9, 23.4, 30.0,63.6, 110.5, 115.8, 124.4, 138.0, 150.0, 160.4,160.6. ESI-MS (NaCl) 605 (2M+Na, 100),314(M+Na,28),292(M+H, 10).Calculated for C₁₃H₁₇N₅OS: C, 53.59; H, 5.88; N, 24.04; S, 11.00. Found:C, 53.43; H, 5.88; N, 24.30; S, 10.86.

Example 13(Z,S)-(+)-2-Amino-6-pentylthio-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(16b)

A mixture of compound 5 (150 mg, 0.6 mmol), potassium carbonate (170 mg,1.2 mmol), 1-pentanethiol (374 mg, 3.60 mmol) and 1,2-dimethoxyethane(10 mL) was stirred at 80° C. for 16 h. The work-up followed theprocedure described in Example 5 to give compound 16b (141 mg, 75%).

Mp. 146–147° C. [α]²⁰ _(D) 82.2° (c=0.37, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 400 MHz) δ0.85 (t, 3H, J=7.2Hz), 1.17–1.21 (m, 1H), 1.25–1.40(m, 4H), 1.45–1.49 (m, 1H), 1;61–1.68 (m, 2H), 2.08–2.12 (m, 1H), 3.25(t, 2H, J=7.4 Hz), 3.28–3.3 (m, 1H), 3.70–3.73 (m, 1H), 5.06 (t, 1H,J=5.2 Hz), 6.57 (bs, 2H), 7.20 (d, 1H, J=1.6 Hz), 8.54 (s, 1H). ¹³C NMR(DMSO-d₆, 100 MHz) ppm 7.0, 14.6, 19.9, 22.4, 28.1, 29.6, 31.1, 63.6,110.5, 115.8, 124.4, 138.0, 150.0, 160.4, 160.6. ESI-MS (NaCl) 661(2M+Na, 20), 639 (2M+H, 28), 320 (M+H, 100). Calculated for C₁₅H₂₁N₅OS:C, 56.40; H, 6.63; N, 21.92; S, 10.04. Found: C, 56.50; H, 6.79; N,21.86; S, 9.96.

Example 14(Z,S)-(+)-2-Amino-6-thio-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine(17)

A mixture of compound 5 (400 mg, 1.6 mmol) and sodium hydrosulfidehydrate (3.00 g, 53.5 mmol) in methanol (50 mL) was stirred at roomtemperature under nitrogen for 24 h The solvent was evaporated, theresidue was dissolved in water (40 mL) and pH of the solution wasadjusted to 6 with acetic acid. The precipitate was filtered off and itwas washed with water. Recrystallization from methanol-water (1:1) gavecompound 17 (265 mg, 67%).

Mp. 240° C. (decomposition). [α]²⁰ _(D) 91.0° (c=0.26,N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 500 MHz) δ1.17–1.21 (m, 1H),1.45–1.49 (m, 1H), 2.08–2.13 (m, 1H), 3.25–3.31 (m, 1H), 3.67–3.72 (m,1H), 5.07 (t, 1H, J=8.0 Hz), 6.86 (s, 2H), 7.09 (d, 1H, J=2.0 Hz), 8.48(s, 1H), 11.96 (s, 1H). ¹³C NMR (DMSO-d₆, 125 MHz) ppm 6.9, 19.8, 63.4,110.5, 116.8, 128.6, 137.9, 146.8, 153.9, 175.7. ESI-MS (NaCl+KCl) 521(2M+Na, 50), 288 (M+K, 20), 272 (M+Na, 100), 250 (M+H, 10). Calculatedfor C₁₀H₁₁N₅OS: C, 48.18; H, 4.45; N, 28.09; S, 12.86. Found: C, 48.10;H, 4.61; N, 28.25; S, 13.02.

Example 15(Z,S)-(+)-2-Amino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine (18)

Compound 17 (166 mg, 0.67 mmol) was dissolved in water-methanol (1:1,100 mL). Raney nickel (approximately 200 mg) was added and the reactionmixture was stirred at room temperature for 16 h. The catalyst wasfiltered off and the filtrate was evaporated. The product waschromatographed using dichloromethane-methanol (9:1) to give compound 18(66 mg, 46%).

Mp. 185–187° C. [α]²⁰ _(D) 77.7° (c=0.30, N,N-dimethylformamide). ¹H NMR(DMSO-d₆, 400 MHz) δ1.19–1.23 (m, 1H), 1.46–1.51 (m, 1H), 2.09–2.16 (m,1H), 3.27–3.33 (m, 1H), 3.70–3.75 (m, 1H), 5.07 (dd, 1H, J=6.2 and 4.6Hz), 6.63 (s, 2H), 7.25 (d, 1H, J=1.6 Hz), 8.60 (s, 1H), 8.68 (s, 1H).¹³C NMR(DMSO-d₆, 100 MHz) ppm 6.9, 19.9, 63.6, 110.3, 115.7, 127.2,140.1, 150.1, 152.0, 161.5. ESI-MS (NaCl) 457 (2M+Na, 25), 240 (M+Na,100), 218 (M+H, 20). Calculated for C₁₀H₁₁N₅O: C, 55.29; H, 5.10; N,32.24. Found: C, 55.40; H, 5.10; N, 32.19.

Example 16 In Vitro Antiviral Evaluation Methods

Cells and viruses. The routine growth and passage of KB cells wasperformed in monolayer cultures using minimal essential medium (MEM)with either Hanks salts [MEM(H)] or Earle salts [MEM(E)] supplementedwith 10% calf serum. The sodium bicarbonate concentration was varied tomeet the buffering capacity required. Cultures of diploid human foreskinfibroblasts (HFF) or MRC-5 cells were grown in medium consisting ofMEM(E) with 10% fetal bovine serum. Cells were passaged at 1:2 to 1:10dilutions according to conventional procedures by using 0.05% trypsinplus 0.02% EDTA in a HEPES buffered salt solution (HBS) (Shipman, C.,Jr., Proc. Soc. Exp. Biol. 130:305–310 (1969)) as described previously.(Turk, S. R., et al., Antimicrob. Agents Chemother. 31:544–550 (1987).HFF and MRC-5 cells were passaged only at 1:2 dilutions.

Virological procedures. Stock HCMV was prepared by infecting HFF cellsat a multiplicity of infection (m.o.i.) of <0.01 plaque-forming units(p.f.u.) per cell. Cell growth medium was changed every four days untilcytopathology was evident in all cells (approximately 21 days).Supernatant fluids were retained as the virus stock. High titer HSV-1stocks were prepared by infecting KB cells at an m.o.i. of <0.1 asdetailed previously. (Turk, S. R., et al., Antimicrob. Agents Chemother.31:544–550 (1987). Virus titers were determined using monolayer culturesof HFF cells for HCMV and monolayer cultures of BSC-1 cells for HSV-1 asdescribed earlier. (Prichard, M. N. et al., J. Virol. Methods 28:101–106(1990). Briefly, HFF or BSC-1 cells were planted as described above in96-well cluster dishes and incubated overnight at 37° C. in a humidified3% CO₂–97% air atmosphere. The next day cultures were inoculated withHCMV or HSV-1 and serially diluted 1:3 across the remaining elevencolumns of the 96-well plate. Cultures were incubated at 37° C. for 2 hrto permit virus adsorption and then virus inoculum was replaced with 0.2mL of fresh medium. Cultures were incubated for seven days for HCMV, twoor three days for HSV-1, medium was removed, and the cell sheets werestained with 0.1% crystal violet in 20% methanol. Plaques wereenumerated under 20-fold magnification in wells having the dilutionwhich gave 5 to 20 plaques per well. Virus titers were calculatedaccording to the following formula: Titer (p.f.u./mL)=number ofplaques×5×3^(n); where n represents the nth dilution of the virus usedto infect the well in which plaques were enumerated.

Assays for Antiviral Activity. (a) HCMV. The effect of compounds on thereplication of HCMV has been measured using a plaque reduction assay.HFF cells in 24-well cluster dishes were infected with approximately 100p.f.u. of HCMV per cm² cell sheet using the procedures detailed above.Following virus adsorption, compounds dissolved in growth medium wereadded to duplicate wells in three to six selected concentrations.Following incubation at 37° C. for 7 to 10 days, cell sheets were fixed,stained with crystal violet and microscopic plaques enumerated asdescribed above. Drug effects were calculated as a percentage ofreduction in number of plaques in the presence of each drugconcentration compared to the number observed in the absence of drug.Ganciclovir (GCV) was used as a positive control in all experiments.

The effect of compounds on the replication of HCMV also was measuredusing a yield reduction assay. HFF cells were planted as described abovein 96-well cluster dishes, incubated overnight, medium removed and thecultures were inoculated with HCMV at a m.o.i. of 0.5 to 1 p.f.u. percell as reported elsewhere. After virus adsorption, inoculum wasreplaced with 0.2 mL of fresh medium containing test compounds. Thefirst row of 12 wells was left undisturbed and served as virus controls.Each well in the second row received an additional 0.1 mL of medium withtest compound at three times the desired final concentration. Thecontents of the 12 wells were mixed by repeated pipetting and thenserially diluted 1:3 along the remaining wells. In this manner, sixcompounds could be tested in duplicate on a single plate withconcentrations from 100 μM to 0.14 μM. Plates were incubated at 37° C.for seven days, subjected to one cycle of freezing and thawing; aliquotsfrom each of the eight wells of a given column were transferred to thefirst column of a fresh 96-well monolayer culture of HFF cells. Contentswere mixed and serially diluted 1:3 across the remaining eleven columnsof the secondary plate. Each column of the original primary plate wasdiluted across a separate plate in this manner. Cultures were incubated,plaques were enumerated, and titers calculated as described above.

Assays for Antiviral Activity. (b) HSV-1. An enzyme-linked immunosorbentassay (ELISA) was employed to detect HSV-1. 96-well cluster dishes wereplanted with BSC-1 cells at 10,000 cells per well, in a total volume of200 μL per well of MEM(E) plus 10% calf serum. After overnightincubation at 37° C., drug and HSV-1 was added at the rate of 100PFU/well. ELISA plates were blocked with 200 μL per well of 10% calfserum and 0.05% tween in HBS. After incubation for 30 minutes, theblocking agent was rinsed two times with HBS-T. A 1:400 dilution of APconjugated rabbit anti-HSV-1 antibody in HBS-F was added. Plates weresealed with adhesive sheet, and incubated on rocker for one hour at 37°C. Plates were developed in the dark with 100 μL per well of substratesolution containing p-nitrophenyl phosphate. Plates were read at 492 nm.Drug effects were calculated as a percentage of the reduction in virusin the presence of each drug concentration compared to the titerobtained in the absence of drug. Acyclovir was used as a positivecontrol in all experiments.

Cytotoxicity assays. Two different assays were used to explorecytotoxicity of selected compounds as we have detailed previously. (i)Cytotoxicity produced in stationary HFF cells was determined bymicroscopic inspection of cells used in plaque assays which were notaffected by the virus. (ii) The effect of compounds during twopopulation doublings of KB cells was determined by crystal violetstaining and spectrophotometric quantitation of dye eluted from stainedcells. (Turk, S. R., et al., Antimicrob. Agents Chemother. 35:1060–1065(1991).

Data Analysis. Dose-response relationships were constructed by linearlyregressing the percent inhibition of parameters derived in the precedingsections against log drug concentrations. Fifty-percent inhibitory(IC₅₀) concentrations were calculated from the regression lines. Samplescontaining positive controls (acyclovir for HSV-1, ganciclovir for HCMV,and 2-acetylpyridine thiosemicarbazone for cytotoxicity) were used inall assays. Results from sets of assays were rejected if inhibition bythe positive control deviated from its mean response by >±1.5 standarddeviations.

Testing Results. The compounds produced in Examples 1–18 exhibit asignificant activity against herpesviruses. It was found that compoundsof the present invention strongly inhibit the replication of HMCV asmeasured by plaque reduction assays using HFF as host cells by themethod described above and they also inhibit the replication of HSV-1 asdetermined by enzyme-linked immunosorbent assay (ELISA).

TABLE 1 Compound IC50 (μM) HCMV^(a) IC50 (μM) HSV-1^(b)  6 1.8 4  7 3.56  8 3.8 2.5  9 3.8 15 10 >100 25 11 >100 15 12 39 >100 13a 0.26 1 13b0.21 7 14 0.33 1.5 15 0.32 7 16a 0.22 1 16b 0.32 7 17 37 >100 18 >100 35Control 4.1^(c) 0.15^(d) ^(a)Plaque reduction assy. ^(b)ELISA.^(c)Ganciclovir. ^(d)Acyclovir.

Compounds of the present invention were also tested for cytotoxicity incultures of HFF and KB cells according to the methods described above.These tests have indicated a complete lack of cytotoxicity of testedcompounds.

TABLE 2 IC₅₀ (μM) IC₅₀ (μM) Compound Stationary HFF cells Growing KBcells  6 >100 >100  7 >100 >100  8 >100 >100  9 >100 >100 10 >100 >10011 >100 >100 12 >100 >100 13a >100 >100 13b >100 >100 14 >100 >10015 >100 >100 16a >100 >100 16b >100 >100 17 >100 >100 18 >100 >100Control  >100^(a)  >100^(b) ^(a)Ganciclovir. ^(b)Acyclovir.

1. A compound having the formula:

wherein B is 2-aminopurine-9-yl, which is unsubstituted or substitutedat the 6 position with NHR₁, OR₂, or SR₃; R₁, is selected from the groupconsisting of alkenyl, alkynyl, and benzyl, any of which may beoptionally substituted with one or more members of the group consistingof hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl and aryl; R₂ isselected from the group consisting of alkenyl, alkynyl, and cycloalkyl,any of which may be branched or unbranched and optionally substitutedwith one or more members of the group consisting of hydroxy, halo,amino, acyl, cycloalkyl, heterocyclyl, and aryl; and R₃ is selected fromthe group consisting of alkyl, alkenyl, alkynyl, and cycloalkyl, any ofwhich may be branched or unbranched and optionally substituted with oneor more members of the group consisting of hydroxy, halo, amino, acyl,cycloalkyl, heterocyclyl and aryl.
 2. The compound of claim 1, wherein Bis 2-aminopurine-9-yl, which is substituted at the 6 position with NHR₁,OR₂, or SR₃; wherein R₁ is selected from the group consisting of C₃–C₈alkenyl, and C₃–C₈ alkynyl, any of which may be optionally substitutedwith one or more members of the group consisting of hydroxy, halo,amino, acyl, cycloalkyl, heterocyclyl and aryl; R₂ is selected from thegroup consisting of C₃–C₈ alkenyl, C₃–C₈ alkynyl, and C₃–C₁₀ cycloalkyl,any of which may be branched or unbranched and optionally substitutedwith one or more members of the group consisting of hydroxy, halo,amino, acyl, cycloalkyl, heterocyclyl and aryl; and R₃ is selected fromthe group consisting of C₂₋₈ alkyl, C₃–C₈ alkenyl, C₃–C₈ alkynyl, andC₃₋₁₀ cycloalkyl, any of which may be branched or unbranched andoptionally substituted with one or more members of the group consistingof hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl and aryl.
 3. Thecompound of claim 1, wherein B is 2-aminopurine-9-yl, which issubstituted in the 6 position with NHR₁, OR₂, or SR₃; wherein R₁ isselected from the group consisting of C₃–C₆ alkenyl, and C₃–C₆ alkynyl,any of which may be optionally substituted with one or more members ofthe group consisting cycloalkyl, heterocyclyl and aryl; R₂ is selectedfrom the group consisting of C₃–C₆ alkenyl, C₃–C₆ alkynyl, and C₃₋₈cycloalkyl, any of which may be branched or unbranched and optionallysubstituted with one or more members of the group consisting ofcycloalkyl, heterocyclyl, and aryl; and R₃ is selected from the groupconsisting of C₂₋₆ alkyl, C₃–C₆ alkenyl, C₃–C₆ alkynyl, and C₃₋₈cycloalkyl, any of which may be branched or unbranched and optionallysubstituted with one or more members of the group consisting of hydroxy,halo, amino, acyl, cycloalkyl, heterocyclyl and aryl.
 4. An antiviralcompound selected from the group consisting of(Z,S)-(+)-2-amino-6-allylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;(Z,S)-(+)-2-amino-6-propargylamino-9-[(2-hydroxymethyl)cyclo-propylidenemethyl]purine;(Z,S)-(+)-2-amino-6-isopropylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;(Z,S)-(+)-2-amino-6-benzylamino9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;(Z,S)-(+)-2-amino-6-cyclohexylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;(Z,S)-(+)-2-amino-6-(2-hydroxy)ethylamino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;(Z,S)-(+)-2-amino-6-allyloxy-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;(Z,S)-(+)-2-amino-6-cyclopropylmethoxy-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;(Z,S)-(+)-2-amino-6-propylthio-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;(Z,S)-(+)-2-amino6-pentylthio-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine;and (Z,S)-(+)-2-amino-9-[(2-hydroxymethyl)cyclopropylidenemethyl]purine.5. A compound having the formula:

wherein X is O; R₁ and R₂ are alkyl or aryl; or R₁X or R₂X is an aminoacid residue with X as NH; B is 2-aminopurine-9-yl, which isunsubstituted or substituted at the 6 position with NHR₄, OR₅, or SR₆;R₄ is selected from the group consisting of alkenyl, and alkynyl, any ofwhich may be optionally substituted with one or more members of thegroup consisting of hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyland aryl; R₅ is selected from the group consisting of alkenyl, alkynyl,and cycloalkyl, any of which may be branched or unbranched andoptionally substituted with one or more members of the group consistingof hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl and aryl; R₆ isselected from the group consisting of alkyl, alkenyl, alkynyl, andcycloalkyl, any of which may be branched or unbranched and optionallysubstituted with one or more members of the group consisting of hydroxy,halo, amino, acyl, cycloalkyl, heterocyclyl and aryl.
 6. The compound ofclaim 5, wherein B is 2-aminopurine-9-yl, which is substituted at the 6position with NHR₄, OR₅, or SR₆; wherein R₄ is selected from the groupconsisting of C₃–C₈ alkenyl, and C₃–C₈ alkynyl, any of which may beoptionally substituted with one or more members of the group consistingof hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl and aryl; R₅ isselected from the group consisting of C₃–C₈ alkenyl, C₃–C₈ alkynyl, andC₃₋₁₀ cycloalkyl, any of which may be branched or unbranched andoptionally substituted with one or more members of the group consistingof hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyl, and aryl; andR₆ is selected from the group consisting of C₂₋₈ alkyl, C₃–C₈ alkenyl,C₃–C₈ alkynyl, and C₃₋₁₀ cycloalkyl, any of which may be branched orunbranched and optionally substituted with one or more members of thegroup consisting of hydroxy, halo, amino, acyl, cycloalkyl, heterocyclyland aryl.
 7. The compound of claim 5, wherein B is 2-aminopurine-9-yl,which is substituted in the 6 position with NHR₄, OR₅, or SR₆; whereinR₄ is selected from the group consisting of C₃–C₆ alkenyl, and C₃–C₆alkynyl, any of which may be optionally substituted with one or moremembers of the group consisting cycloalkyl, heterocyclyl and aryl; R₅ isselected from the group consisting of C₃–C₆ alkenyl, C₃–C₆ alkynyl, andC₃₋₈ cycloalkyl, any of which may be branched or unbranched andoptionally substituted with one or more members of the group consistingof cycloalkyl, heterocyclyl, and aryl; and R₆ is selected from the groupconsisting of C₂₋₆ alkyl, C₃–C₆ alkenyl, C₃–C₆ alkynyl, and C₃₋₈cycloalkyl, any of which may be branched or unbranched and optionallysubstituted with one or more members of the group consisting of hydroxy,halo, amino, acyl, cycloalkyl, heterocyclyl and aryl.
 8. A compositioncomprising a compound of any one of claims 1 to 7 and a pharmaceuticallyacceptable carrier.